Biology subjects

Hernaez, M. L.

Publications and source records attributed to Hernaez, M. L..

5 recordsLinked to original sources

bioRxiv2026

Proteomics to uncover new actors in the antifungal action of metformin. Impact on virulence traits, oxidative stress, and essential proteins from Candida albicans.

Metformin is one of the most widely prescribed drugs with a proven safety profile, making it an excellent candidate for drug repurposing strategies. Its anti-Candida effect and its synergistic potential with azoles have already been described against C. albicans and C. glabrata, alongside AMPK involvement and its divergent consequences on autophagy. However, metformin has additional effects on C. albicans biology that remain to be explored using differential proteomics. In the present study, C. albicans SC5314 was treated with metformin to identify the proteins involved in its anti-Candida activity. First, we demonstrated that metformin inhibits C. albicans SC5314 growth in a dose-dependent manner, an effect that intensifies under low glucose (0.2%) and filament-inducing conditions. It also reduces virulence traits such as filamentation, biofilm formation, and invasive growth. Furthermore, it induces significant oxidative stress, which is neutralized by antioxidant agents such as N-acetylcysteine and glutathione. Additionally, 50 mM metformin substantially increased fluconazole efficacy under 0.2% glucose. Our label-free proteomic study of C. albicans SC5314 exposed to 50 mM metformin allowed the identification and quantification of 1,899 proteins, with 95 and 47 proteins showing increased and decreased abundance, respectively. Notably, 26 of the down-regulated proteins were encoded by essential genes, demonstrating the drastic effect of metformin on C. albicans viability. GO Term analysis revealed that the most relevant functions affected were ATP binding, inhibition of ATPase activity (with reduced ATP levels), translation inhibition, mitochondrial function, filamentation, and responses to oxidative stress and antifungals. In conclusion, metformin significantly compromises the viability and virulence of C. albicans at high concentrations. Given that high concentrations of metformin have recently been reported in the intestinal tract, and that intestinal Candida is a known source of invasive infections in immunocompromised, under chemotherapy, and post-surgery patients, understanding metformin anti-Candida action becomes increasingly relevant.

microbiology↗

bioRxiv2026

A generalizable normalization framework to decouple protocol and instrument effects: Application to high-sensitivity proteomics multicentric study (PME13)

Multicenter studies are essential for benchmarking analytical workflows, yet their interpretation is often confounded by the combined effects of experimental protocols and instrumentation. To address this challenge, we introduce a simple normalization-based analytical framework, the recovery metric ({rho}), designed to decouple protocol driven effects from instrument dependent variability. We applied this framework to the 13th Proteomics Multicentric Experiment (PME13), a large multicentric proteomics dataset generated across 27 laboratories using high sensitivity workflows and varying sample preparation protocols. By leveraging a common digested reference sample, {rho} enables direct cross-comparison of all datasets on a unified scale, effectively minimizing instrument-related biases. Using this approach, we demonstrate that apparent instrument dependent trends are largely removed when evaluated through {rho}, revealing consistent protocol driven effects across laboratories. Statistical modeling identified key variables influencing {rho}, including sample input amount, reduction and alkylation, and the use of n-dodecyl-{beta}-D-maltoside (DDM). While DDM was associated with improved {rho}, reduction and alkylation and additional handling steps led to reduced performance, particularly at low input levels. We further highlight practical considerations for the application of ratio based normalization, including the occurrence of values exceeding theoretical bounds, which reflect deviations from underlying assumptions and require appropriate filtering. Overall, this work establishes a generalizable analytical strategy for disentangling confounding factors in multicentric datasets and provides practical guidelines for optimizing high sensitivity proteomics (HSP) workflows. The proposed framework is broadly applicable to other analytical fields where cross laboratory comparability is required.

bioinformatics↗

bioRxiv2023

Unravelling the Role of Candida albicans Prn1 in the Oxidative Stress Response Through a Proteomic Approach

Candida albicans Prn1 is a protein that shares similarities with mammalian Pirin but with an unknown function in the yeast. Orthologues of Prn1 have been identified in other pathogenic fungi but not in Saccharomyces cerevisiae, suggesting a relationship with pathogenesis. Prn1 increase in abundance after H2O2 treatment has been shown previously, thus, in the present work, C. albicans prn1{Delta} mutant and the corresponding wild-type strain SN250 have been treated with H2O2 and their response was studied by quantitative differential proteomics. These assays indicated a lower increase of proteins with oxidoreductase activity after treatment in the prn1{Delta} strain compared to the wild type, as well as an increase in proteasome-activating proteins and a decrease in translation-involved proteins. Accordingly, Prn1 absence, under H2O2 treatment, led to a lower survival rate and a higher percentage of apoptosis, together with higher reactive oxygen species levels and higher proteasome activity. Besides, remarkable differences in the abundance of some transcription factors were observed between the two strains. Mnl1, involved in Prn1 expression, Bas1, Tiff33, and orf19.1150 presented an inverse pattern of expression under H2O2 treatment respect to Nrg1, a Mnl1 antagonist. Interestingly, orf19.4850, a protein orthologue to S. cerevisiae Cub1, has shown to be involved in the response to H2O2 presenting a conserved proteasome function. Under basal conditions, the proteomics results indicate a possible involvement of Prn1 in mitochondrial oxidative stress detoxication. Our experiments confirm Prn1 as a relevant actor in the oxidative response. ImportanceCandida albicans is a human opportunistic pathogen included in the WHO fungal priority pathogens list. The increase in resistant strains necessitates the discovery of new targets for antifungal therapies. Our research sheds light on the important role of the previously uncharacterized C. albicans protein Prn1 during the oxidative stress response. Study of the proteome remodelling under oxidative stress unveils the role of Prn1 in the decreased reactive oxygen species levels and the consequences, such as death by apoptosis and necrosis or cell growth delay. A proteomics approach allowed the identification of several proteins potentially involved in Prn1 activity, such as oxidoreductases and transcription factors. The lack of Prn1 orthologues in Saccharomyces cerevisiae but the presence in other Candida and Aspergillus species implicates this protein in pathogenesis and suggests that it may serve as a candidate for new drug targets.

microbiology↗

bioRxiv2020

Distinct human gut microbial taxonomic signatures uncovered with different sample processing and microbial cell disruption methods for metaproteomic analysis

Metaproteomics is as a promising technique for studying the human gut microbiota, because it can reveal the taxonomic profile and also shed light on the functional role of the microbial community. Nevertheless, methods for extracting proteins from stool samples continue to evolve, in the pursuit of optimal protocols for moistening and dispersing the stool sample and for disrupting microbial cells which are two critical steps for ensuring good protein recovery. Here, we evaluated different stool sample processing and microbial cell disruption methods for metaproteomic analyses of human gut microbiota. An unsupervised principal component analysis showed that different methods produced similar human gut microbial taxonomic profiles. An unsupervised two-way hierarchical clustering analysis identified the microbial taxonomic signatures associated with each method. Proteobacteria and Bacteroidetes identification was favored by moistening the stool samples during processing and by disrupting cells with medium-sized glass beads. Ascomycota identification was enhanced by using large-sized glass beads during sample processing for stool dispersion. Euryarchaeota identification was improved with a combination of small and medium-sized glass beads for cell disruption. Assessments of the relative abundance of Firmicutes, Actinobacteria and Spirochaetes improved when ultrasonication was performed before cell disruption with glass beads. The latter method also increased the overall number of identified proteins. Taxonomic and protein functional analyses of metaproteomic data derived from stool samples from six healthy individuals showed common taxonomic profiles. We also detected certain proteins involved in microbial functions relevant to the host and related mostly to particular taxa, such as B12 biosynthesis and short chain fatty acid production carried out mainly by members in the Prevotella genus and the Firmicutes phylum, respectively. Finally, in this metaproteomic study we identified several human proteins, mostly related to the anti-microbial response, which could contribute to determining the beneficial and detrimental relationships between gut microbiota and human cells in particular human diseases.

microbiology↗

bioRxiv2020

Small extracellular vesicles secreted by Candida albicans hyphae have highly diverse protein cargoes that include virulence factors and stimulate macrophages

Extracellular vesicles (EVs) have been described as mediators of microorganism survival and interaction with the host. In Candida albicans, a relevant commensal fungal pathogen, the dimorphic transition is an important virulence trait in candidiasis. We have analyzed EVs secreted by yeast (YEVs) or hyphal cells (HEVs) from C. albicans, finding interesting differences in both size distribution and protein loading. In general, HEVs were smaller and carried a much more diverse protein cargo than YEVs, including most of the proteins identified in YEVs, which were mainly cell surface proteins. Virulence factors such as phospholipases, aspartic proteases (Saps), adhesins and invasins, and the precursor protein of candidalysin toxin Ece1p were also detected. HEVs were also enriched in proteasomal and ribosomal proteins, and in enzymes from amino acid biosynthetic pathways, all involved in protein metabolism, as well as proteins related to intracellular protein transport and components of the ESCRT pathway related to exosome biogenesis. Both types of EV presented immune reactivity with human sera from patients suffering invasive candidiasis. In our conditions, only HEVs could elicit the release of TNF by activated macrophages. This first analysis of C. albicans HEVs shows their relevance to pathogenesis and possible new diagnostics or treatments.

microbiology↗